A CXC chemokine sequence isolated from the rainbow trout Oncorhynchus mykiss resembles the closely related interferon-gamma-inducible chemokines CXCL9, CXCL10 and CXCL11

A sequence encoding a CXC - type chemokine from rainbow trout was found to most resemble members of the CXCL9/CXCL10/CXCL11 sub-family. In mammals, all 3 chemokines are regulated by IFN-gamma and are chemotactic for activated T lymphocytes. The trout chemokine (gammaIP1), with a message of 787 nucleotides, contains 100 amino acids in a typical non-ELR CXC chemokine arrangement. A second sequence (gammaIP2), with 6 nucleotide differences in the coding region when compared to the first, was also identified although it is not known whether this is a second functional gene or a second allele. The gene is separated onto 4 exons, and the introns intervene in conserved positions according to the mammalian equivalents. The sequence encoded by the second exon shares the highest amino acid identity (37%) with CXCL10, with lower values of identity to other CXC chemokines (17-31%). Furthermore, phylogenetic analysis groups the trout chemokine with mammalian CXCL9, CXCL10 and CXCL11 peptides. Constitutive expression of gammaIP is seen in trout gill and low level expression in spleen, head kidney and liver. In RTS-11 cells, gammaIP expression can be induced with poly I:C, but not by LPS, suggesting virus-mediated regulation of gammaIP. Intraperitoneal injection of recombinant trout TNF-alpha caused elevation in gammaIP mRNA levels in trout head kidney.     

The transfer of purine metabolites via the medium rather than through cell contacts

When the medium in which mouse B82 cells had been grown for 24 h with [³H]hypoxanthine was given to HGPRT⁻ Chinese hamster cells (CHW-1102), the acid-insoluble fraction of these cells became radioactive. When the medium in which mouse B82 cells had been grown for 24 h without [³H]hypoxanthine was given to CHW-1102 cells at the same time as [³H]hypoxanthine was added, the acid-insoluble fraction of the cells did not become radioactive. This indicates that CHW-1102 cells acquire from the B82 medium ³H material and not the ability to utilize hypoxanthine. Very little radioactivity was found in the acid-insoluble fraction of the B82 medium and when the medium was given to CHW-1102 cytoplasms, they did not become labelled. These results suggest that ³H purine metabolites (and not ³H nucleic acids) are responsible for the radioactivity in the CHW-1102 cells. Such ³H metabolites were also present in the medium of mouse L929 cells, but were absent in the medium of Chinese hamster (V79), mouse (A9), Syrian baby hamster kidney (BHK) and human fibroblasts. The cells were judged to be free of mycoplasma by three different criteria. This exchange of metabolites through the medium is referred to as contact-independent metabolite transfer (CIMT).     

Synthesis and deconvolution of the first combinatorial library of glycosidase inhibitors.

A combinatorial library of 125 compounds with a structure consisting of 1-azafagomine linked at N-1 via an acetic acid linker to a variable tripeptide was synthesised. The library was synthesised by Merrifield split and mix synthesis of the peptide, followed by capping with chloroacetate, regioselective nucleophilic substitution with 1-azafagomine and cleavage from the polymeric support. The library was screened for inhibition of beta-glucosidase, alpha-glucosidase and glycogen phosphorylase and found to display beta-glucosidase inhibition. Deconvolution of the library revealed that some inhibition was caused by all library members but the strongest inhibitor was clearly a compound having three hydroxyproline residues in the peptide fragment. This compound was a weaker but more selective inhibitor than 1-azafagomine itself.     

Effects of aspiration vacuum and needle diameter on cumulus oocyte complex morphology and developmental capacity of bovine oocytes

The effects of aspiration vacuum and needle diameter on the morphology of the cumulusoocyte-complex (COC) and developmental capacity of the oocyte after IVF was studied in 2 experiments using a disposable ovum pick-up needle guidance system whose construction permits its use in vitro. In Experiment 1, the relationship was determined between the aspiration vacuum, expressed in millimetre of mercury, and the actual amount of water aspirated by the system, expressed in millilitre per minute. In Experiment 2, five different levels of aspiration vacuum for 3 different needle diameters (18g, 19g and 21g) were tested in slaughterhouse ovaries. The cumulus-oocyte complexes (COCs) were divided into 3 categories: 1) oocytes with a compact cumulus, 2) oocytes with an expanded cumulus and 3) naked oocytes. The results show that a change of needle diameter can triple the amount of fluid actually aspirated. The highest oocyte recovery rates are obtained when using the thickest needle (18-g), regardless of the aspiration vacuum. On the average, for all needle types, more oocytes are recovered at the highest aspiration vacuum. For all needle diameters, the proportion of oocytes surrounded by a compact cumulus decreases progressively as the vacuum increases. Regardless of the vacuum applied, thinner needles result in a higher proportion of recovered COCs with a compact cumulus. At a high aspiration vacuum, naked oocytes become predominant regardless of the needle diameter. The prevalence of blastocysts, expressed in proportion to the recovered COCs, decreases as the aspiration vacuum increases, being especially noticeable between 70 and 130 mm Hg.     

Relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained placenta

A study was conducted to investigate the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in cows that experienced a dystocia or retained their placenta. Fifteen healthy cows, 31 cows with retained placenta (RP) and 13 cows that had dystocia were clinically examined 1 or 2 days after parturition when a uterine swab for bacteriological examination was taken. In addition, plasma and uterine lochia samples were collected to determine lipopolysaccharide (LPS) and the plasma IgG anti-LPS concentrations. Subsequently, 15 RP and 6 dystocia cows were initially left untreated and another uterine swab was collected at 2 and 4 wk postpartum. Immediately after calving, RP cows had significantly higher LPS levels in uterine lochia (average of 2.24 x 10(4) Endotoxin Units (EU)/mL) as compared to dystocia and healthy postpartum cows (average of 0.10 and 0.26 EU/mL, respectively). However, plasma LPS levels were below the detection limit (<0.036 EU/mL platelet-rich plasma) in all groups of cows. IgG anti-LPS levels in plasma were not significantly different between the 3 groups immediately postpartum (average of 26, 16 and 44 Median Units (MU)/mL) for healthy, dystocia and RP cows, respectively), but they were significantly lower when compared to plasma IgG anti-LPS levels of healthy cows at more than 2 months postpartum (mean 83 MU/mL). High LPS levels in lochia at 1 or 2 days postpartum were significantly related to abnormal cervical discharge, the presence of Escherichia coli, black pigmented gram-negative anaerobes and Clostridium spp. shortly after calving, and Arcanobacterium pyogenes and gram-negative anaerobes in the uterus at 14 days postpartum. These results suggest that the presence of E. coli and LPS (endotoxins) in lochia early postpartum favor the development of uterine infections by A. pyogenes and gram-negative anaerobes later postpartum. LPS were not observed in plasma, suggesting that either they are not absorbed into the blood, or they are efficiently detoxified by IgG anti-LPS or other detoxification mechanisms.     

Characterisation of low, medium and high responders following FSH stimulation prior to ultrasound-guided transvaginal oocyte retrieval in cows

In human IVF, the concept of 'low responders' is well known and generally defined as women with poor-response to gonadotrophin stimulation in a previous induction cycle. The objective of this retrospective study is to describe and characterise the concepts of 'low-, medium-, and high-response' and 'low, medium, and high responders' in bovine-assisted reproduction by analysing the OPU-IVF results obtained following 665 gonadotrophin-stimulated sessions conducted in 112 animals, nearly all of which were previously unsuccessful in traditional multiple ovulation and embryo transfer (MOET) programs. They were submitted to OPU and IVP between 1999 and 2003. In reference to these 665 OPU sessions, categories of response were defined based on the overall mean+/-S.D. follicles aspirated and COC obtained i.e., for follicles 14.7+/-9.8 and for COCs 11.7+/-8.1. So arbitrary cut-off values to define the categories of sessions were for follicles 5 and 25, and for COC 4 and 20. The three categories for follicles punctured in one session were therefore follicle low-response (FLR)or=25 follicles and for COCs recovered in one session COC low-response (CLR)or=20 COC. In addition, four categories of animals were also defined: (1) a low responder animal (LRA) had at least one OPU session in which FLR and CLR were observed (genuine low-response, see Section ); these animals did not have any high-response sessions, (2) a medium responder animal (MeRA) had only medium-responses, (3) a high responder animal (HRA) had at least one OPU session in which FHR and CHR were observed; these animals did not have any low-response sessions, and (4) mixed responder animals (MiRA) had both low and high-responses. Finally, we distinguished biological (animals) and technical (recovery rate and ultrasound resolution) causes of response differences. In 'low, high, medium and mixed reponders,' different results were obtained (p<0.05): mean follicle numbers (8.8+/-4.8a, 22.4+/-10.5c, 13.2+/-5.2b,15.1+/-10.2d), COC numbers (6.3+/-3.9a, 18.5+/-8.2c, 10.4+/-4b, 12.0+/-8.3d), embryo numbers (1.8+/-2.1a, 5.6+/-4.9c, 2.5+/-2.7b, 3.5+/-3.8d) and also for recovery rate (72%a, 83%b,79%, 79%) and percentage embryo development (29%, 30%a, 24%b, 29%). In conclusion, the results of this study demonstrate that variability in OPU results has technical (ultrasound resolution and recovery rate) as well as biological (animal) aspects. Selection of animals with extreme (high or low) follicle and COC production results allows us to distinguish three populations: 'low, medium, and high responders' to FSH stimulation.     

Slow inhibition of almond beta-glucosidase by azasugars: determination of activation energies for slow binding

The thermodynamic and activation energies of the slow inhibition of almond beta-glucosidase with a series of azasugars were determined. The inhibitors studied were isofagomine ((3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 1), isogalactofagomine ((3R,4S,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine, 2), (-)-1-azafagomine ((3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine, 3), 3-amino-3-deoxy-1-azafagomine (4) and 1-deoxynojirimycin (5). It was found that the binding of 1 to the enzyme has an activation enthalpy of 56.1 kJ/mol and an activation entropy of 25.8 J/molK. The dissociation of the enzyme-1 complex had an activation enthalpy of -2.5 kJ/mol and an activation entropy of -297 J/molK. It is suggested that the activation enthalpy of association is due to the breaking of bonds to water, while the large negative activation entropy of dissociation is due at least in part to the resolvation of the enzyme with water molecules. For the association of 1 DeltaH(0) is 58.6 kJ/mol and DeltaS(0) is 323.8 J/molK. Inhibitor 3 has an activation enthalpy of 39.3 kJ/mol and an activation entropy of -17.9 J/molK for binding to the enzyme, and an activation enthalpy of 40.8 kJ/mol and an activation entropy of -141.0 J/molK for dissociation of the enzyme-inhibitor complex. For the association of 3 DeltaH(0) is -1.5 kJ/mol and DeltaS(0) is 123.1 J/molK. Inhibitor 5 is not a slow inhibitor, but its DeltaH(0) and DeltaS(0) of association are -30 kJ/mol and -13.1 J/molK. The large difference in DeltaS(0) of association of the different inhibitors suggests that the anomeric nitrogen atom of inhibitors 1-4 is involved in an interaction that results in a large entropy increase.     

Induction of 7-ethoxyresorufin-O-deethylase activity by planar chlorinated hydrocarbons and polycyclic aromatic hydrocarbons in cell lines from the rainbow trout pituitary

The induction of 7-ethoxyresorufin-o-deethylase (EROD) activity was examined in three rainbow trout pituitary cell lines: RTP-91E, RTP-91F and RTP-2. RTP-91E and RTP-91F were developed from the pituitary of a male and have epithelial-like and fibroblast-like morphologies, respectively. RTP-2, which was described previously, was developed from the pituitary of a female and has an epithelial-like shape. In all cell lines EROD activity was induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Immunoblotting with the polyclonal antibody, anti-trout CYP1A1(277-294)/KLH, confirmed induction of a 58-kDa polypeptide. Potential inhibitors of the aryl hydrocarbon receptor, geldanamycin and alpha-naphthoflavone, inhibited EROD induction by TCDD. Other compounds inducing EROD activity were 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3-methylcholanthrene (3MC). When judged by the concentration eliciting 50% of the maximal response (EC50), induction was similar in RTP-2 and RTP-91E, and less effective in RTP-91F. Regardless of the cell line, the rank order from most to least potent inducer on the basis of EC50 value was TCDD> or =PCDD>TCDF>PCB 126>3MC. When induction potencies were expressed relative to TCDD, the values obtained with the pituitary cell lines were similar to previously published values derived with a rainbow trout liver cell line.     

Enzyme inhibition by iminosugars: Analysis and insight into the glycosidase–iminosugar dependency of pH

Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme–inhibitor complexes with three (EH₃I), two (EH₂I), one (EHI), or no protons (EI), are possible. In the present work an analysis method is presented that from pH-inhibition data allows one to distinguish between the different complexes and determine which protonation state is preferred. It is also possible to determine the pH-independent binding constants of the inhibitor. Analysis of pH data for imino- and azasugar inhibition of β-glucosidases revealed that basic glycosidase inhibitors bind as the monoprotonated (EHI) complex. Three neutral inhibitors were also studied and two of these were also bound exclusively as the EHI complex while a third bound both as a EHI and a EH₂I complex.